How We Work
There are three general ways that user labs interface with us:
Collaborative Project
The core facility lab becomes your collaborator. We get intellectually invested in your project as well as provide mentoring and supervision to your staff that will be working in the core facility. We will work alongside and mentor your personnel, help them with experimental design and analysis, as well as aid your lab with grant writing and manuscript preparation.
Extension of your Lab
The core facility is setup so that an experienced scientist can come in and operate independently. If you have appropriately experienced staff they are free to use the facility to conduct their research, so long as the facility director is confident in their abilities. In all likelihood some limited training will be necessary on our equipment and operations.
Independent Project
In the event that you do not have the staff and/or the interest the core facility has all the expertise and equipment necessary to independently tackle your structural biology project (or some aspects of it) for you.
At any time you can adjust how you interface with us, our mission is to help you accomplish your research goals in whatever way is best for you.
Frequently asked questions
How much protein is needed?
At least 1 mg concentrated to 10-15 mg/ml.
In more detail, crystallization is attempted in screens of 96 different reagent solutions. Each solution is placed in proximity to a drop composed of 100 nl protein plus 100 nl solution. That means each screen is 100 nl * 96 = 9.6 ul, but we account for a little loss during pipetting such small volumes, so round up to 12 ul protein per screen. We want to do several screens at once, anywhere from 4-10, so we can call that around 100 ul protein at minimum. The protein must be fairly highly concentrated to get it to the range where crystallization can occur. That’s generally recommended to start at 10-15 mg/ml, then we go up or down based on observations. A 100 ul sample at 10 mg/ml would be just 1 mg. More is better — it would allow for a higher protein concentration and more screening.
How pure must the sample be?
We need the sample to be as pure as reasonably achievable. Often we will make sacrifices in the yield of a prep in order to get a purer product. In addition, the molecule of interest should neither aggregate nor degrade, and it should be monodisperse in solution. We prefer to assess dispersity and oligomeric state using mass photometry.
How much does it cost?
Crystallization tests would be around $2-5K. Then getting diffraction data and determining the structure would be around $5-10K.
Can you handle custom ligands?
Yes, definitely. Depending on the ligand’s nature, solubility, size, and binding affinity to the protein of interest, we may prefer to pursue a cocrystallization strategy or one of crystallizing the apo-enzyme then soaking in the ligand.
How long does it take?
The whole process could take anywhere from 3-12 months. Sometimes we get lucky and everything goes quickly and smoothly. Other times we get mired in one or another stage – usually trying to get crystals, but sometimes dealing with mediocre data or crystal pathologies.
Rigor and Reproducibility
Rigor and reproducibility are crucial to your research aims, and they are becoming greater factors in securing critical grant funding opportunities. There are eight steps to Rigorous and Reproducible Experiments in Biomolecular Research at UNC:
1. If using a core facility, consult with the core staff in the planning stage. Consult with a statistician if you need help developing a Power Analysis to assure that your results will be adequately powered.
2. Design your experiment with sufficient controls (rigor) and replicates (reproducibility).
3. Assure that ALL of your reagents (antibodies, cell lines, mice) are fully validated (see below).
4. Have a clear and detailed protocol (SOP) and data analysis plan that can be easily followed. Assure that the protocol is strictly followed or that any deviation is well documented.
5. Assure that the staff or students performing the experiment are well trained and understand each step and the importance of performing them precisely (rigor again).
6. Use only well-maintained instrumentation, preferably maintained and operated in a core facility with expert staff (see #1 above).
7. Document all steps, reagents, equipment and data analysis methods used in the experiment. Assure that the both the documentation and the data itself are properly stored in a safe data management repository.
8. Acknowledge the Cancer Center Support Grant (P30 CA016086), Grant-supported instrumentation and core staff in publications.
You may use this as a template for wording in your grants and publications to address Rigor and Reproducibility for your research proteins generated by the the UNC Protein Expression and Purification Core Facility. We can also provide statements to this effect in Letters of Support for your grant applications.